Project Summary: Hypoxia and ischemia (HI) related brain injury after perinatal asphyxia is a major cause of death and life-long disability, occurring in 20,000 newborns every year in the United States. Female newborns are two times more resistant to the effects of perinatal asphyxia, a phenomenon that is poorly understood. Our recent studies have demonstrated a similar female bias in neuroprotection following neonatal HI in mice, and have clearly implicated estrogen receptor alpha (ERa) in conferring this sex-specific neuroprotective mechanism. We recently reported that ERa upregulation confers neuroprotection in the female neonatal hippocampus through cross-talk with the neurotrophin receptor, tyrosine kinase B (TrkB), which has been shown to play an important role in neuroprotection and improving long-term functional recovery in neonatal mice post-HI. Administration of 7,8-dihydroxyflavone (7,8-DHF; potent and selective TrkB agonist) increases TrkB phosphorylation and hippocampal neuronal survival post-HI in female, but not in male newborn mice, and these female specific effects are absent in the ERa null mutant mice. In preliminary experiments we have found that female-specific upregulation of hippocampal ERa post-HI is dependent on a demethylating DNA repair factor, the growth arrest and DNA damage-inducible protein 45 beta (Gadd45b). Additionally, when we treated females with testosterone (T) during the perinatal period, subsequent HI-induced expression of hippocampal ERa is reduced to male levels. In the proposed experiments, we will determine the mechanisms by which sex-specific ERa expression and neuroprotection occurs following neonatal HI, and interrogate signaling pathways that mediate ERa cross-talk with TrkB and thereby confer its neuroprotective effects. We will test the following aims in mice and sexed hippocampal neurons following in vivo and in vitro ischemia, respectively: In Aim 1, we will interrogate whether membrane ERa signaling mediates neuroprotection post-HI, and assess signaling mechanisms that link ERa signaling to the membrane TrkB receptor. In Aim 2, we will interrogate if knockdown Gadd45b alters the methylation status of the ERa gene promoter region in a female specific manner following in vitro ischemia. In Aim 3, we will evaluate the effects of altered perinatal testosterone exposure on ERa expression and the severity of long-term functional deficits. The work proposed in these aims will test the novel hypothesis that sex-specific TrkB phosphorylation is mediated by HI induced ERa upregulation. An improved understanding of the mechanisms underlying sex-based susceptibility to HI will provide new avenues for future development of drug therapies for neonatal HI.